The success of allogeneic organ transplantation has been established in the last few decades, but the limited supply of donor organs means that many patients have little or no chance of receiving a transplanted organ, such as a kidney, heart or liver. A significant number of these people die whilst awaiting an organ. One potential solution is “xenografting”, or the use of organs from a non-human (“xenogeneic”) animal donor.
Porcine donor organs are thought to be suitable candidates because pigs are anatomically and physiologically similar to humans and are in abundant supply. Porcine organs are rejected rapidly upon revascularisation, however, by a humoral process called hyperacute rejection (HAR). This is caused by naturally-occurring antibodies in the recipient which recognise and cross-react with antigens on the endothelial cells (ECs) of the xenograft. This recognition triggers the complement cascade which in turn leads to rejection.
European patent 0495852 (Imutran) suggests that membrane-bound regulators of host complement should be expressed on the xenograft in order to prevent the complete activation of complement in the organ recipient. This approach has been developed and applied in order to produce transgenic animals with organs designed to survive hyperacute rejection [eg. refs 1 & 2].
However, organs surviving HAR are subject to delayed xenograft rejection (DXR). This is characterised by the infiltration of recipient inflammatory cells and thrombosis of graft vessels, leading to ischaemia. WO98/42850 shows that expression of coagulation inhibitors on the surface of the xenograft can inhibit the thrombotic aspect of this type of rejection.
HAR and DXR are followed by the host T lymphocyte-mediated response. There are two pathways, “direct” and “indirect” by which T-cells may become sensitised against xenoantigens. The direct pathway involves interactions between T-cells and MHC molecules on xenogeneic donor cells, whereas the indirect pathway involves the presentation of processed xenoantigens by host APCs in the context of MHC class II. The indirect T-cell response is much stronger against xenoantigens than against alloantigens [3], which contrasts with findings for the direct pathway [4], indicating that both the direct and indirect human T-cell responses against xenoantigens must be suppressed if xenotransplantation is to be effective.
It appears that the suppression of anti-xenograft indirect T-cell responses will be one of the greatest challenges for xenotransplantation [5,6]. Maintaining the level of immunosuppression required to prevent chronic xenograft rejection due to persistent indirect immunogenicity may be unfeasible using conventional systemic immunosuppressive drugs because of the increased the risks of infection and neoplasia [eg. 7]. Clearly, if xenotransplantation is to be clinically successful, methods to promote graft-specific immunosuppression are needed in order to reduce the requirements for systemic therapy.
T-cell activation requires two separate signals. Delivery of signal 1 alone induces a refractory state (“anergy”), defined as the inability to produce IL-2 after subsequent antigenic exposure. For full activation to occur, the cell must be co-stimulated with signal 2.
In vivo, signal 1 is provided by the interaction of the TCR/CD4 complex with either allogeneic MHC or antigenic peptide complexed with self MHC; signal 2 is supplied by the interaction between B7 molecules (B7.1 and B7.2, also known as CD80 and CD86, respectively) on the antigen-presenting cell (APC) and CD28 on the T-cell.
Monoclonal antibodies (mAbs) have played a key role in studying T-cell activation. Signal 1 can be supplied by mAbs directed against the TCR/CD3 complex, and mAbs against CD28 can provide signal 2. Indeed, T-cells can be activated by two suitable mAbs, even in the absence of APC. Activation can also be prevented, rather than provided, using mAbs. Signal 2 can be blocked, for instance, using mAbs which block either B7 or CD28.
Signal 2 can also be blocked by using modified forms of CTLA-4, a high-affinity ligand for B7. CTLA-4 is a natural negative regulator of T-cell activation, and B7 binding to CTLA-4 on an activated T-cell antagonises the co-stimulatory signal provided by the B7/CD28 interaction. Soluble forms of CTLA-4, consisting of the extracellular domains of CTLA-4 linked to the constant domain of an antibody, have been constructed [8,9] to block T-cell activation. These molecules (“CTLA4-Ig” or “CTLA4-Fc”) behave in a similar way to anti-B7 antibodies and have been used in vitro and in vivo to prevent the co-stimulatory functions of B7 and thus promote tolerance [10].
Targeting the B7/CD28 interaction to prevent T cell sensitisation to graft antigens in vivo has been shown to be an effective strategy to enhance graft survival. Using CTLA4-Ig, prolonged survival has been obtained in various allograft models [eg. 11] and in a human-to-murine islet xenograft model [12]. In the xenograft model, CTLA4-Ig administration caused full tolerance against the xenoantigens by rendering direct-reactive T cells anergic.
It is thus an object of the invention to provide means to promote xenograft-specific immunosuppression. In particular, it as an object of the invention to inhibit T-cell-mediated rejection of xenotransplanted organs by preventing the organ recipient's T-cells from mounting an immune response against the organ. More specifically, it is an object to prevent this immune response by inducing anergy in the recipient's T-cells which recognise the xenotransplanted organ, resulting in xenograft-specific T-cell tolerance.